Collagen is an abundant protein found in the connective tissues of animals. Procollagen undergoes a number of post-translational modifications which enhance stability, folding, and function. Two of the modifications involve hydroxylation of proline, which requires selective reaction with an unactivated C-H bond, either at the 4-(R)- or 3-(R)-position. Such reactions are catalyzed by the enzymes prolyl 4- hydroxylase (P4H) and prolyl 3-hydroxylase (P3H), respectively. Prolyl hydroxylases are targets for inhibition in the treatment of fibrotic diseases and severe scarring, conditions associated with excessive collagen formation. Information about substrate binding geometry and the mechanism of this reaction is lacking, in part because the crystal structures of these proteins have not been solved. The objective of this project is to develop a better understanding of the mechanism of hydroxylation through the use of small organic molecules as mechanistic probes. It is hypothesized that the proline amide bond of the peptide substrate is in the cis-conformation in the active site of the enzyme. The requirement for cis-peptide bond conformation would provide a means to prevent product inhibition and impart selectivity, as the resulting hydroxyproline products, as well as other amino acids, have significantly greater preference for the trans- peptide bond over the cis-peptide bond conformation than proline itself. To test this hypothesis, it is proposed to synthesize E- and Z-alkene peptide analogues of procollagen designed to mimic the trans- and cis-conformations of the amide bond of proline. The research strategy is focused on four specific isosteres designed as substrates for P4H and P3H, which will be analyzed to determine the effect of conformation on the ability to bind and react with each enzyme. Understanding of substrate conformational requirements in the enzyme active site will provide insight about the mechanism of hydroxylation. Furthermore, information about substrate binding geometry should inform the design of selective inhibitors of prolyl hydroxylases for the treatment of fibrotic diseases. PUBLIC HEALTH RELEVANCE: The proposed research aims to investigate the reaction mechanism of enzymes that catalyze a posttranslational modification required for the production of collagen. Insight into the mechanism should inform the future design of inhibitors, as these enzymes are seen as potential targets for the treatment of fibrotic diseases and severe scarring, conditions characterized by excessive collagen formation. Understanding gained from the project may also be applicable to related enzymes that are involved in sensing molecular oxygen, which could be targeted for the treatment of ischemia, stroke, or myocardial infarction.